Enzyme Cascades controlled in the Electrochemical Leaf for Discovery in Antimicrobial Strategy and Novel Bio-electrochemical Systems

This project will exploit the Electrochemical Leaf (e-Leaf) (a new concept/platform in which tandem catalysis by multi-enzyme cascades is electrochemically driven and controlled) to study specific bacterial metabolic and bioenergetic pathways with separate aims: to discover novel antimicrobial strategies and to create new electrochemical platform technologies for the control of enzyme systems that are currently unexplored by direct electrochemistry.

Antibiotic discovery is usually aimed at single entities, for example a bacterial enzyme or single efflux protein. This also means that antimicrobial resistance (AMR) mechanisms are considered in terms of the individual response, for example, mutations in a single target enzyme, affording resistance to the drug. But the inherent synergy between the multi-enzyme cascades of bacterial metabolism offers a new way to target bacteria.

One aim of this project is to discover a new strategy aimed at targeting this enzyme teamwork in bacterial metabolism to lead to their attenuation.

It is currently impossible to insightfully monitor multi-enzyme pathways and their "teamwork" in vitro in real-time and as such, novel ways to target this synergy in bacteria are unexplored. The problem is that typical in vitro enzyme investigations are passive (no way to drive) and take place in dilute solution; there is no convenient in vitro technique that provides a way to drive the catalysis of these enzyme teams and to observe their catalytic activity in real-time and therefore their 'live' response to other metabolites or small molecule inhibitors.

Moreover, it is impossible to monitor their activity in vitro under conditions like those in which they function in vivo - nanoconfined and highly crowded cellular compartments.

The ability to use electrochemistry to monitor enzyme teams would give unique insight since it brings thermodynamic control to kinetics, and is interactive, but multi-enzyme cascades which contain enzymes from different classes other than redox, are not amenable to direct electrochemical control. The solution is the e-Leaf, in which the crowded nanoconfinement of enzymes in a porous metal oxide electrode, and a key enzyme in photosynthesis called ferredoxin NADP+ reductase (FNR) to transduce electricity to connect to an extended cascade, now enables these multi-enzyme pathways to be electrochemically driven and controlled under conditions that mirror those in vivo.

As such, the unique insight electrochemistry gives, can now be exploited for this untapped area in AMR and the student will initially focus on the folate pathway which is targeted by sulfonamides and diaminopyrimidine and is subject to regulation by metal ions and allosteric effects. It is ideal for this fundamental study - both the bacterial folate pathway and the human folate pathway can connect to FNR in the e-Leaf through dihydrofolate reductase.

In addition to this focus, the student will electrochemically study other native pathways from specific anaerobic bacterial strains. Enzyme engineering will be incorporated into the project and overall applications include antimicrobial strategies and new bio-electrochemical systems for synthesis.

This project falls within several of the UKRI's research priorities including Antimicrobial Resistance, Biophysics, Energy, Engineering, Manufacturing the Future (Building a Green Future), Chemical Biology and Biological chemistry, Electrochemical Sciences, and Catalysis.